PROJECT SUMMARY/ABSTRACT Currently, 8-10 million people in United States suffer from vascular disease, and a large number of those affected succumb to death and major amputation. Development of methods to recreate microvascular networks and increase perfusion is necessary to prevent tissue damage. Mesenchymal stem cells (MSCs) derived from a patient?s bone marrow or adipose tissue are increasingly utilized in the clinic as a result of their potential to secrete multiple pro-vascular and regenerative factors. These factors include vascular endothelial growth factor, plasminogen activator inhibitor, and matrix metalloproteinase-9. However, it is a challenging task to sustainably regulate the secretory activity within an ischemic tissue. The goals of this proposed project are therefore to develop a nanostimulator that can associate with MSCs and regulate stem cell stromal capacity, and to use it to improve angiogenesis and physiological outcomes. We hypothesize that a nanostimulator assembled to sustainably deliver insoluble and soluble signals, two of which separately involve in cellular secretome, can sustainably stimulate MSCs to release therapeutic molecules in the ischemic tissue. To test this hypothesis, we will use interferon-gamma (IFN?) as a soluble stimulatory factor and CD44-binding hyaluronic acid (HA) as an insoluble stimulatory factor. These molecules will be spatially organized in the 400 nm-diameter poly(ethylene glycol)diacrylate gel/liposome core/shell nanoparticles. We will accomplish our goal by first modifying the core/shell particle surface with HA and examining the extent to which the resulting nanoparticle associates with MSCs and regulates cellular secretion activities (Aim 1). Second, we will introduce IFN? into the core/shell nanoparticles and evaluate the extent to which the sustained molecular release modulates cellular secretion activities (Aim 2). Finally, we will determine the extent to which MSCs loaded with the optimized core/shell nanostimulator can promote reperfusion of ischemic muscle and minimize tissue damage using a mouse ischemic hindlimb model (Aim 3). The significance of this study lies in the ability to (1) enhance the therapeutic capacity of MSCs and (2) improve quality of ischemic muscular treatment using a reduced number of stem cells. Accordingly, the innovation components include (1) control of the nanostimulator stability in physiological condition, (2) spatial organization of the HA and IFN? in the nanoparticle, (3) in situ orthogonal tailoring of cell?s secretory activities, and (4) the multidisciplinary team established to conduct this study (Kong, PI, biomaterials; Boppart, Co-I, stem cell and muscle biology).